Derivation and Characterization of EGFP-Labeled Rabbit Limbal Mesenchymal Stem Cells and Their Potential for Research in Regenerative Ophthalmology

Abstract

The development of cell-based approaches to the treatment of various cornea pathologies, including limbal stem cell deficiency (LSCD), is an area of current interest in regenerative biomedicine. In this context, the shortage of donor material is urgent, and limbal mesenchymal stem cells (L-MSCs) may become a promising cell source for the development of these novel approaches, being established mainly within the rabbit model. In this study, we obtained and characterized rabbit L-MSCs and modified them with lentiviral transduction to express the green fluorescent protein EGFP (L-MSCs-EGFP). L-MSCs and L-MSCs-EGFP express not only stem cell markers specific for mesenchymal stem cells but also ABCG2, ABCB5, ALDH3A1, PAX6, and p63a specific for limbal epithelial stem cells (LESCs), as well as various cytokeratins (3/12, 15, 19). L-MSCs-EGFP have been proven to differentiate into adipogenic, osteogenic, and chondrogenic directions, as well as to transdifferentiate into epithelial cells. The possibility of using L-MSCs-EGFP to study the biocompatibility of various scaffolds developed to treat corneal pathologies was demonstrated. L-MSCs-EGFP may become a useful tool for studying regenerative processes occurring during the treatment of various corneal pathologies, including LSCD, with the use of cell-based technologies.

Improving the properties of fish skin collagen/silk fibroin dressing by chemical treatment for corneal wound healing

“…CK12 expression is restricted to the corneal epithelium. 80 Antibody staining for vimentin revealed long straight fibrils in mesenchymal cells, while vimentin was found as a dense network of thin fibres in epithelial differentiation, 81 as observed in our study. Most HCECs on the hydrogel membranes and TCPS groups were positive for putative stem cell marker CK19 as well as differentiation cell markers (CK3 and CK12); also, some cells expressed ABCG2 (putative limbal epithelial stem cells), specific patterned expression of vimentin (epithelial differentiation marker), according to immunocytochemistry analysis (corneal and limbal supra‐basal epithelial cells marker).…”

Labial Mucosa Stem Cells: Isolation, Characterization, and Their Potential for Corneal Epithelial Reconstruction

“…Mesenchymal stem cells of different origins are known to be able to express both epithelial and mesenchymal markers cultured under certain conditions and to be induced to differentiate into epithelial way. 39 – 41 For instance, Ma et al successfully used bone marrow-derived mesenchymal stem cells to restore chemically damaged rat corneas. 42 Thus, the co-expression of epithelial and mesenchymal markers in both cultures of rLMSCs and hLMSCs is consistent with the understanding that mesenchymal stem cells could be induced to differentiate into epithelial way and indicates their potential for corneal epithelial reconstruction.…”

Corneal Reconstruction with EGFP-Labelled Limbal Mesenchymal Stem Cells in a Rabbit Model of Limbal Stem Cell Deficiency

“…Recently, we demonstrated that rabbit L-MSCs had a high proliferative potential in vitro, expressed epithelial markers, and could differentiate into epithelial-like cells [ 22 ]. The ability of MSCs of various origins to differentiate in diverse directions has been widely discussed in the literature [ 14 , 17 , 18 , 26 , 27 , 28 , 29 ].…”

“…This study aimed to assess the fate of L-MSCs-EGFP [ 22 ] after transplantation onto a damaged cornea in the LSCD model in rabbits. Cells were transferred as part of a tissue-engineered graft based on dHAM.…”

“…Therefore L-MSCs seem more effective in corneal epithelial repair [ 6 , 19 ]. L-MSCs have been shown to express similar stemness markers to LESCs, such as ABCG2, ABCB5, PAX6, and p63a [ 4 , 11 , 20 , 21 , 22 ]. The plasticity of these cells and the possibility to differentiate into various directions have been previously shown [ 23 ].…”